The present invention relates to the use of binding partners for 5-HT5 receptors for the treatment of neuropathological disorders and associated indications, symptoms and dysfunctions and to processes for the identification and characterization of binding partners of this type.
At least seven different receptor classes mediate the manifold physiological activities which are ascribed to an involvement of the neurotransmitter serotonin (5-hydroxytryptamine, abbreviated 5-HT). According to an internationally recognized classification, they are designated by 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. Most of these classes moreover include receptor types which can be differentiated further. Thus the 5-HT1 class includes receptors which can be divided into at least five subclasses, which are designated by 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess F. G. and Martin I. L., Neuropharmacology 33:275-317 (1994)).
The 5-HT5 class was described for the first time by Plassat et al., The EMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992). 5-HT5A and 5-HT5B receptors are differentiated (Erlander et al., Proc. Natl. Acad. Sci. USA 90:3452-3456 (1993)). Only small sequence homologies exist between 5-HT5 and other 5-HT receptors. The pharmacological profile of these receptors differs markedly. Using molecular biology techniques, the localization of 5-HT5 receptors was possible in the olfactory bulb, in the hippocampus, in the cortex, in the cerebral ventricles, in the corpus callosum and in the cerebellum. By means of immunohistochemical methods, it was possible to show that 5-HT5 receptors are principally expressed on astrocytes (Carson et al., GLIA 17:317-326 (1996)). Astrocytes are directly adjacent to the basal membrane of brain capillaries of the blood-brain barrier. An abnormal astrocyte endothelium structure accompanies a loss of the blood-brain barrier. The exact significance of the astrocytes is unclear. They appear to look after transport tasks and connective functions. Reactive astrocytes were observed in connection with reactive gliosis in a number of pathological brain changes and neuropsychiatric disorders. As a result of brain injuries, they change their morphologies. The protein expression pattern changes and growth factors are produced. In vitro investigations on cultured astrocytes have allowed the detection of 5-HT5 receptor-mediated responses. It is thus to be suspected on the one hand that they are involved in recovery processes of the brain after disorders, but on the other hand it is also not to be excluded that they contribute to the creation of damage or even to an increase in damage.
CNS disorders nowadays concern large sections of the population. In particular on account of the increase in elderly people, the numbers of patients are increasing continuously. Neuropathological conditions such as cerebral ischemia, stroke, epilepsy and attacks in general, chronic schizophrenia, other psychotic disorders, dementia, in particular Alzheimer""s dementia, demyelinizing disorders, in particular multiple sclerosis, and brain tumors lead to damage to the brain and the neuronal deficits associated therewith.
Therapeutic treatments of the neurodegenerative and neuropsychiatric disorders outlined were up to now directed at various membrane receptors with the aim of compensating deficits in neurotransmission processes. Indeed, it was possible to achieve neuroprotective effects with serotonogic compounds in animal models of neuropathological conditions, such as ischemia, cerebral stroke and excitotoxicity. In some cases, it was also possible to observe favorable effects on emotional disturbances, such as depression or anxiety states. Mention may be made here, for example, of 5-HT1A agonists, such as buspirone or the compound 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), which is characterized as a selective 5-HT1A receptor ligand. These active compounds, however, only decrease neurological deficits to a limited extent. There is still no effective therapy at present.
It is therefore an object of the present invention to make possible the treatment of neuropathological disorders with adequate efficacy and minor side effects.
Surprisingly, it has now been found that treatment of the above disease conditions and associated indications, symptoms and dysfunctions is made possible by specific use of substances having binding affinities for 5-HT5 receptors.
One subject of the present invention is therefore the use of binding partners for 5-HT5 receptors for the preparation of an agent for the treatment of neuropathological disorders and associated indications, symptoms and dysfunctions.
Neuropathological disorders are understood according to the invention as meaning disorders which are accompanied by neurological deficits, i.e. a condition characterized by neurological deficiency symptoms. The term xe2x80x9cdisorderxe2x80x9d in the sense according to the invention designates anomalies which, as a rule, are regarded as pathological conditions and can reveal themselves in the form of certain signs, symptoms and/or dysfunctions. The treatment according to the invention can be directed at individual disorders, viz. anomalies or pathological conditions, but a number of anomalies which are causally connected to one another can be combined to give patterns, i.e. syndromes, which can be treated according to the invention.
This condition can exist temporarily, progressively or persistently.
According to the invention, the treatment of neurodegenerative and/or neuropsychiatric disorders is preferred. These disorders occur, in particular, in neuropathological syndromes, as a rule syndromes caused by brain damage, for example cerebral ischemia, stroke, epilepsy and seizures in general, chronic schizophrenia, other psychotic disorders, dementia, in particular Alzheimer dementia, demyelinizing disorders, in particular multiple sclerosis, and brain tumors. The invention in particular also relates to the use of 5-HT5 binding partners for the treatment of those forms of the abovementioned disorders in whose formation and/or course 5-HT5 receptors are involved, i.e. disorders which are modulated by a 5-HT5 receptor activity.
According to a further aspect of the present invention, neuropathological disorders are treated which accompany a glial reaction. The use according to the invention relates in particular to the modulation of a glial reaction.
An advantageous action of the binding partners is seen in the preventive or acute treatment of neurological deficits, which are observed in patients who suffer from psychiatric disorders, such as epilepsy, psychosis, e.g. psychoses of the acute exogenous reaction type or concomitant psychoses of organic or exogenous cause, e.g. after trauma, especially brain lesions and diffuse brain damage, in metabolic disorders, infections, and endocrinopathies; endogenous psychoses, such as schizophrenia, and schizotypic and delusional disorders; effective disorders, such as depression, mania and manic depressive conditions; and mixed forms of the psychoses described above; senile dementia and senile dementia of the Alzheimer type, and in the treatment or prevention of demyelinization processes.
The binding partners according to the invention are efficacious, in particular with respect to the treatment of ischemic damage, e.g. as a result of brain and spinal cord trauma and vascular occlusion or heart failure.
Especially to be mentioned here is stroke (synonym: cerebral apoplexy, cerebral or apoplectic insult, cerebral stroke). Transitory ischemic attacks, reversible ischemic neurological deficits, prolonged reversible ischemic neurological deficits, partially reversible ischemic neurological symptoms and also persistent complete cerebral infarcts can be treated according to the invention. The treatment of acute forms is particularly advantageous according to the invention.
One or more of the changes in nerve tissues listed below underlie the forms of neuropathological disorders which can be preferably treated according to the invention: degeneration or death of neurons, in particular of the ganglial cells, e.g. tigrolysis, indistinctness of the nuclear membrane, plasmolysis, cytoplasm vacuolization and encrustation, parenchymal necroses of the brain, cerebral edema, changes to neurons caused by oxygen deficiency, atrophy, morphological changes, such as demyelinization, in particular medullary sheath disintegration, perivascular infiltrates, glial proliferation and/or glial scarring; degeneration of the Substantia nigra.
The indication to be treated according to the invention is often characterized by a progressive development, i.e. the conditions described above change in the course of time, as a rule the degree of severity increases and conditions further to already existing conditions can occur.
By means of the treatment according to the invention of neuropathological disorders or of the conditions underlying them, a number of further signs, symptoms and/or dysfunctions which are connected with the neuropathological disorders can be treated, i.e. in particular accompany the disorder conditions described above. These include, for example, shock lung, brain nerve losses, e.g. retrobulbar neuritis, eye muscle paralysis, syllabication, spastic paralysis, cerebella symptoms, sensitivity, bladder and rectal disorders, euphoria, dementia, hyper- and akinesia, absence synchynesis, small-step gait, bent posture of trunk and limbs, pro-, retro- and lateropulsion, tremor, lack of facial expression, monotonous speech, depression, apathy, labile or rigid affectivity, impaired spontaneity and resoluteness, slowed thinking, poor association ability; muscular atrophy.
A treatment in the sense according to the invention comprises not only the treatment of acute or chronic signs, symptoms and/or dysfunctions but also a preventive treatment (prophylaxis), in particular as a relapse or phase prophylaxis. The treatment can be achieved symptomatically, for example as symptom suppression. It can be carried out short-term, be carried our medium-term, or it can also be a long-term treatment, for example in the context of a maintenance therapy.
The term xe2x80x9cbinding partner for 5-HT5 receptorsxe2x80x9d describes substances which bind to 5-HT5 receptors and can therefore also be designated as 5-HT5 receptor ligands.
Binding is understood as meaning any molecular interaction between the binding partner and the receptor, in particular under physiological conditions. As a rule, these are conventional interactions, which include electrostatic attraction, hydrogen bonding, hydrophobic bonds, van-der-Waals forces or metal complex-like coordinative bonds. In addition to the abovementioned, reversible molecular interactions, irreversible interactions between binding partner and receptor can also be possible, such as, for example, covalent bonds.
According to one embodiment, binding partners which can be used according to the invention competitively inhibit the binding of comparison binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine), to 5-HT5 receptors.
Competitive inhibition is understood as meaning that binding partners which can be used according to the invention compete with a comparison binding partner, in the present case, for example, 5-HT or 5-CT, for binding to the receptor, i.e. the binding of one prevents the binding of the other.
According to a further embodiment, binding partners which can be used according to the invention inhibit the binding of comparison binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine) to 5-HT5 receptors noncompetitively.
Noncompetitive inhibition is understood as meaning that binding partners which can be used according to the invention modulate, via their binding to the receptor, the binding of a comparison binding partner, in the present case, for example, 5-HT or 5-CT, in particular lower its binding affinity.
At least in the case of competitive inhibition, i.e. of reversible binding, the principle applies that the displacement of one binding partner by another increases with decreasing binding affinity of the one or increasing binding affinity of the other with respect to the receptor. More expediently, therefore, binding partners which can be used according to the invention have a high binding affinity for 5-HT5 receptors. A binding affinity of this type allows, on the one hand, an effective displacement of naturally occurring binding partners for 5-HT5 receptors, such as, for example, serotonin (5-hydroxytryptamine, 5-HT) itself, where the necessary concentration of binding partner which can be used according to the invention for the binding of a certain amount of this binding partner to 5-HT5 receptors decreases with increasing binding affinity. With respect to medical use, binding partners are therefore preferred whose binding affinity is so great that these can be administered in justifiable amounts in the course of an effective medical treatment as an active compound. Binding partners which can be used according to the invention are therefore preferably administered in daily doses of approximately 0.01 to 100 mg/kg of body weight and in particular of approximately 0.1 to 15 mg/kg of body weight on parenteral administration and 1 to 30 mg/kg of body weight on oral administration.
The competition experiments referred to above, with which that concentration of binding partner which can be used according to the invention is determined in vitro which displaces 50% of another comparison binding partner from the receptor binding site (IC50 values), offer one possibility of expressing the binding affinity. Thus the competitive inhibition of the binding of 5-CT to 5-HT5 receptors can also be evaluated to the effect that binding partners which can preferably be used according to the invention have half-maximal inhibition constants IC50 of less than 10xe2x88x925 M, preferably of less than 10xe2x88x926 M and in particular of less than 10xe2x88x927 M.
The binding affinity of binding partners which can be used according to the invention can also be expressed by means of the inhibition constant Ki, which is in general likewise determined in vitro using competition experiments. For the binding of 5-HT5 receptors, binding partners which can be used according to the invention preferably have Ki values of less than 10xe2x88x926 M, advantageously of less than 10xe2x88x927 M and particularly preferably of less than 10xe2x88x928 M. Ki values of compounds which can be used according to the invention lie, for example, in the range from 1xc2x710xe2x88x927 M to 7xc2x710xe2x88x927 M or in the range from 1xc2x710xe2x88x928 M to 1xc2x710xe2x88x927 M.
Binding partners which can be used can bind with a lower, an essentially identical, or a higher affinity to 5-HT5 than to a specific receptor which is different from 5-HT5.
Thus binding partners for 5-HT5 receptors with respect to the use according to the invention in particular include those whose binding affinity for 5-HT5 receptors compared with the affinity for 5-HT1 receptors, in particular 5-HT1A, 5-HT1B and/or 5-HT1D, is so high that they are advantageously suitable for the use according to the invention. This does not necessarily presuppose a comparatively more selective binding to 5-HT5 receptors, even though selective binding partners for 5-HT5 receptors are a particular embodiment of the present invention.
For example, binding partners can be used which have high affinity both for 5-HT5 and for 5-HT1 receptors, in particular for 5-HT1A, 5-HT1B and/or 5-HT1D. In this connection, high affinity means Ki values as a rule in the range from 1xc2x710xe2x88x929 M to 1xc2x710xe2x88x926 M. According to a particular embodiment, binding partners which can be used in the high affinity range have a binding profile for 5-HT receptors which is characterized by a binding affinity to 5-HT5 which, in comparison to other binding affinities of this range is essentially identical or only slightly less. Factors of 10 or less can be advantageous.
Selective binding partners which can be used according to the invention have binding affinities for 5-HT5 receptors which are larger than for one or more 5-HT receptors which are different from 5-HT5, i.e. in particular receptors allocated to the abovementioned 5-HT receptor classes 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT6 and 5-HT7. If the binding affinity for 5-HT5 receptors of a binding partner is greater than that of a 5-HT receptor which is different from 5-HT5, we speak of a selective binding of these binding partners to 5-HT5 receptors in relationship to the 5-HT receptor which is different from 5-HT5. Particular binding partners are those whose binding affinity for 5-HT5 receptors is greater than for at least one and in particular all 5-HT1 receptors, in particular for 5-HT1A, 5-HT1D and/or 5-HT1B receptors. Binding partners whose binding affinity for 5-HT5 receptors is greater than for all 5-HT receptors which are different from 5-HT5 constitute a further particular class of binding partners according to the invention.
Selectivity is understood as meaning the property of a binding partner to bind preferably to 5-HT5 receptors.
It is decisive for the selectivity outlined above that the binding affinities for 5-HT5 receptors on the one hand and for one or more of 5-HT receptors which are different from 5-HT5 on the other hand are adequately different. Affinity differences are preferred according to which binding affinity ratios of at least 2, advantageously of at least 5, particularly advantageously of at least 10, preferably of at least 20, particularly preferably of at least 50 and in particular of at least 100 are present.
According to a further embodiment, binding partners which can be used according to the invention bind selectively to 5-HT5 receptors having the advantageous binding affinities described above in relation to one or more 5-HT receptors other than 5-HT5.
According to a further embodiment, binding partners which can be used according to the invention bind selectively to 5-HT5 receptors having the advantageous binding affinities described above in relation to all 5-HT receptors other than 5-HT5.
Binding partners are particularly advantageous which bind to 5-HT5 receptors which are expressed by glia cells and in particular by astrocytes with the affinities and selectivities described above.
According to the invention, the human receptor variant is a preferred target for the binding partners employed according to the invention.
The binding of binding partners according to the invention to 5-HT5 receptors is coupled to an effector function. Binding partners can act agonistically or antagonistically and partly agonistically and/or partly antagonistically.
Agonists are designated as compounds according to the invention which completely or partially imitate the activity of 5-HT on 5-HT5 receptors.
Antagonists are designated as compounds according to the invention which can block the agonistic activity of 5-HT on 5-HT5 receptors.
According to a preferred embodiment of the present invention, binding partners are employed whose binding at least to 5-HT5 receptors of h5-HT5-transfected CHO cells brings about a change in the agonist-induced stimulation of GTP binding to membrane-bound G proteins, a change in intracellular calcium levels, a change in agonist-induced induction of phospholipase C activity and/or a change in cAMP production. As far as the change in intracellular calcium levels is concerned, the use of binding partners which bring about an increase in intracellular calcium levels represents a particular embodiment of the invention.
This embodiment also includes binding partners which are active in known animal models for neurodegenerative and neuropsychiatric processes.
Preferred binding partners are those which are also selective for 5-HT5 receptors in relation to their effector function in the sense described above.
Compounds which can be used according to the invention are described, for example, in DE 197 24 979.5. These are 3-substituted 3,4,5,6,7,8-hexahydropyrido[3xe2x80x2, 4xe2x80x2:4,5]thieno-[2,3-d]pyrimidine derivatives of the formula I 
in which
R1 is a hydrogen atom, a C1-C4-alkyl group, an acetyl group, a phenylalkyl C1-C4 radical, where the aromatic is optionally substituted by halogen, C1-C4-alkyl, trifluoromethyl, hydroxyl, C1-C4-alkoxy, amino, cyano or nitro groups or is a phenylalkanone radical, where the phenyl group can be substituted by halogen,
R2 is a phenyl, pyridyl, pyrimidinyl or pyrazinyl group, which is optionally mono- or disubstituted by halogen atoms, C1-C4-alkyl, trifluoromethyl, trifluoromethoxy, hydroxyl, C1-C4-alkoxy, amino, monomethylamino, dimethylamino, cyano or nitro groups, and which can optionally be fused to a benzene nucleus, which can optionally be mono- or disubstituted by halogen atoms, C1-C4-alkyl, hydroxyl, trifluoromethyl, C1-C4-alkoxy, amino, cyano or nitro groups and can optionally contain 1 nitrogen atom, or to a 5- or 6-membered ring which can contain 1-2 oxygen atoms,
A is NH or an oxygen atom,
Y is CH2, CH2xe2x80x94CH2, CH2xe2x80x94CH2xe2x80x94CH2 or CH2xe2x80x94CH,
Z is a nitrogen atom, carbon atom or CH, where the bond between Y and Z can also be a double bond,
and n is the number 2, 3 or 4
and their salts with physiologically tolerable acids.
Further compounds which can be used according to the invention are described, for example, in DE 196 36 769.7. These are 3-substituted 3,4,5,6,7,8-hexahydropyrido[4xe2x80x2,3xe2x80x2:4,5]thieno-[2,3-d]pyrimidine derivatives of the formula I 
in which
R1 is a hydrogen atom, a C1-C4-alkyl group, an acetyl or benzoyl group, a phenyl C1-C4-alkyl radical, where the aromatic is optionally substituted by halogen, C1-C4-alkyl, trifluoromethyl, hydroxyl, C1-C4-alkoxy, amino, cyano or nitro groups, or is a naphthyl-C1-C3-alkyl radical, a phenyl-C2-C3-alkanone radical or a phenylcarbamoyl-C2-alkyl radical, where the phenyl group can be substituted by halogen,
R2 is a phenyl, pyridyl, pyrimidinyl or pyrazinyl group, which is optionally mono-, di- or trisubstituted by halogen atoms, C1-C4-alkyl, trif luoromethyl, trif luoromethoxy, hydroxyl, C1-C4-alkoxy, amino, monomethylamino, dimethylamino, cyano or nitro groups, and which can optionally be fused to a benzene nucleus which can optionally be mono- or disubstituted by halogen atoms, C1-C4-alkyl, hydroxyl, trifluoromethyl, C1-C4-alkoxy, amino, cyano or nitro groups and can optionally contain 1 nitrogen atom, or to a 5- or 6-membered ring which can contain 1-2 oxygen atoms,
or can be substituted by a phenyl-C1-C2-alkyl or -alkoxy group, where the phenyl radical can be substituted by halogen, or a methyl, trifluoromethyl or methoxy group,
A is NH or an oxygen atom,
B is hydrogen or methyl,
C is hydrogen, methyl or hydroxyl,
X is a nitrogen atom,
Y is CH2, CH2xe2x80x94CH2, CH2xe2x80x94CH2xe2x80x94CH2 or CH2xe2x80x94CH,
Z is a nitrogen atom, carbon atom or CH, where the bond between Y and Z can also be a double bond,
and n is the number 2, 3 or 4,
and their salts with physiologically tolerable acids.
5-HT5-specific antibodies can also be utilizable as 5-HT5 binding partners. They can be polyclonal antisera, monoclonal antibodies, antibody fragments, such as F(ab), Fc, etc., chimeric and recombinant antibodies. Such antibodies can be prepared in a manner known per se. As an immunogen, 5-HT5 receptor can be used as such or antigenic fragments thereof, as a rule coupled to customary carrier proteins.
Further low molecular weight 5-HT5 binding partners, usually synthetic compounds, can be used advantageously in many respects.
Aptamers, i.e. nucleic acids, as a rule oligonucleotides, having sufficient affinity for 5-HT5 receptors, can also be used as binding partners.
The use according to the invention is not restricted to the abovementioned binding partners. Rather, any substance which binds to 5-HT5 receptors in the manner described above, can be used according to the invention as a 5-HT5 binding partner.
Assays for the determination of binding affinities of test substances for 5-HT5 receptors are known in principle. This can be carried out, for example, by assessing the competitive inhibition of the binding of a comparison binding partner to 5-HT5 receptors by the substance to be investigated. Suitable comparison binding partners are known ligands for 5-HT receptors, such as 5-HT or 5-CT or LSD. These are expediently labeled such that their binding to 5-HT receptors can be monitored analytically using standard methods. Radioactive and optical markers are preferred. In binding studies on 5-HT5 receptors, according to the invention, 5-CT or LSD, in particular in the form of [3H]-LSD, is used. The binding affinities can be expressed as half-maximal inhibition constants IC50 or as inhibition constants Ki. This process is preferably used for primary screening. SPA technology or FlashPlate technology is preferably used.
The binding to binding partners to be investigated can also be determined directly on 5-HT receptors. The inhibition constants Kiexpressing binding affinity can be determined, for example, calorimetrically, i.e. by measurement of the binding energy released.
Effector functions can also be assessed qualitatively or quantitatively both in vitro and in vivo with the aid of known functional assays.
The assessment of an agonistic activity can be based on all those effects which are produced by the binding of 5-HT to 5-HT5 receptors. It is preferred according to the invention to assess the effects on the binding of GTP to G proteins, on intercellular calcium levels, on the phospholipase C activity and/or on the cAMP production. These processes are preferably used for secondary screening. Here too, SPA or FlashPlate technology is advantageously used.
GTP binding to G proteins can be investigated by using a nonhydrolyzable analog of GTP, for example [35S]GTPxcex3S, whose binding can be investigated radiologically. This investigation is preferably carried out on membranes having 5-HT5 receptors.
For the measurement of intracellular calcium levels, it is possible to employ suitable calcium probes, as a rule calcium chelating agents, for example fluorescing compounds, such as Fura 2-acetylmethyl ester or fluo-3-AM. This investigation is preferably carried out on cell cultures having 5-HT5 receptors, in particular on individual cells.
The phospholipase C activity can be determined by means of the reactions catalyzed by it, for example, the incorporation of myoinositol, which for detection purposes is preferably radiolabeled as [3H]-myoinositol, or the conversion of PPIP2 to IP3, where the PPIP2 is also preferably radiolabeled as [32P]PIP2. These investigations are preferably carried out on individual cells having 5-HT5 receptors.
cAMP production can be determined with the aid of the cAMP binding protein. This investigation is preferably carried out on individual cells having 5-HT5 receptors.
If appropriate, the effector function is also determined, i.e. the activity of binding partners according to the invention for other 5-HT receptors. This expediently takes place taking into account the binding affinities determined for 5-HT5 and other 5-HT receptors, i.e. in particular taking into account the selectivity.
The present invention therefore also relates to processes for the identification and characterization of binding partners which can be used according to the invention. These and further processes which are similarly suitable can form the basis for in vitro screening processes with which it is possible from a large number of different compounds to pick out those which, with respect to future use, appear to be the most promising. For example, by means of combinatorial chemistry, extensive substance banks can be prepared which comprise myriads of potential active compounds. The inspection of combinatorial substance libraries for substances having desired activity is automatable. Screening robots are used for the efficient evaluation of the individual assays, which are preferably arranged on microtiter plates. Thus the present invention also relates to screening processes, i.e. both primary and secondary screening processes, in which preferably at least one of the processes described below is used. If a number of processes are used, this can be shifted in terms of time or simultaneously carried out on one and the same sample or on different samples of a substance to be investigated.
A particularly effective technology for carrying out processes of this type is the scintillation proximity assay, called SPA for short, known in the field of active compound screening. Kits and components for carrying out this assay can be obtained commercially, for example from Amersham Pharmacia Biotech. In principle, solubilized or membrane-bound receptors are immobilized on small fluoromicrospheres containing scintillation substance. If, for example, a radioligand binds to the immobilized receptors, the scintillation substance is stimulated to emit light, since the spatial vicinity between scintillation substance and radioligand is specified.
A further particularly effective technology for carrying out processes of this type is the FlashPlate(copyright) technology known in the field of active compound screening. Kits and components for carrying out this assay can be obtained commercially, for example from NEN(copyright) Life Science Products. This principle is likewise based on microtiter plates (96-well or 384-well), which are coated with scintillation substance.
The abovementioned assays are known in principle to the person skilled in the art.